Method for automatically matching serial cross-sections observed with a microscope

ABSTRACT

Three holes in known relative positions are made in a sample to be examined. After the sample has been serially cross-sectioned, the sections are imaged. Movement and deformation of each section is determined by comparing the positions of the holes in the sections to that of the original known relative positions. Based on the changes, an inverse transformation is determined and applied to the section image. The transformed images then provide serial cross sectional images of the sample without the degradation produced by the sectioning and imaging process.

BACKGROUND OF THE INVENTION

This invention concerns a process that makes it possible to automate thecalculation of deformations that make possible the matching ofsuccessive sections of microscopy, electronic transmission microscopyand optical microscopy.

The matching of successive sections is traditionally done by movingimages according to the operator's judgment. This method does not makeit possible to correct the deformations that the mechanical action ofthe cutting introduces to the thin sections.

Some deformations are related to the techniques used to make it possibleto visualize successive sections in microscopy. These include

Translation and rotation related to:

placement of the sections one after the other within the grids orsupporting blades which cannot be reproduced exactly, and

deformations related to the mechanical actions of the microtome or theultramicrotome.

Also included is

Systematic rotation related to different powers of enlargement (in thecase of an electron microscope in transmission mode).

SUMMARY OF THE INVENTION

The process of the invention makes it possible to correct all thesedeformations by calculating an inverse transformation function andestablishing a fixed marker derived from markings previously obtainedmechanically or by firing a pulsed laser.

The process according to the invention takes place after thehistological preparation stage, prior to observation with the opticalmicroscope or the electron transmission microscope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a process according to the invention.

FIG. 2 is a perspective view of marking holes in a microscopicpreparation (weak magnification).

FIG. 3 is a plan view of an example of an electron microscope grid witha series of sections.

FIGS. 4A-4C are schematic drawings of the acquisition of images androtation related to the functioning of an electronic transmissionmicroscope between two different magnifications.

FIGS. 5A-5B are schematic drawings of deformations related to sectioningtechniques.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The process according to the invention includes several steps: (See FIG.1)

1. "Marking"

The process according to the invention includes a sample preparationstep before a series of sections is cut (see FIG. 2). The microscopicpreparation 1 is pierced by at least three holes 2 not in a straightline near area to be studied and made either mechanically or by means ofa pulsed laser. The barycenters of these holes 2 will be used as markingpoints to establish a fixed marker and for final calculation of theinverse transformation function.

2. "Section in Series"

The process according to the invention takes the classic step of makingsuccessive sections 4 of the preparation 1 using a microtome for thesections to be viewed by optical microscopy and an ultramicrotome forsections to be viewed with an electron microscope (see FIG. 3).

3. Digitation

The process according to the invention includes observation steps withthe optical microscope or with the electron transmission microscope, aswell as the acquisition and digitation of images by a computer system.This computerized system includes a camera and an image-digitationsystem connected to a computer. Three images are acquired for an imageto be studied (see FIG. 4):

Image A: The image that is going to be studied.

Image B: An image obtained at a weaker magnification, without moving theslide 3 making it possible to see at least one of the marking holes 2.

Image C: An image making it possible to visualize all the marking holes2, at the same magnification as for image B.

4. "Normalization"

A normalization step for digital processing of all the images A of thesections to be studied, making it possible to obtain homogeneousinformation in terms of contrast and distribution of grey levels.

The distributions of the grey levels of the images are normalized interms of their average and their variance.

Enhancement is done by dispersion of the diagram distributions.

Specific mathematical functions are used for the work on thedistributions (improvement and stabilization).

5. "Calculation of the Inverse Transformation"

The process according to the invention includes a step for calculatingthe inverse transformation function making it possible to re-establishmore closely from an initial section considered a reference a set markerthat is fixed for all the sections. This method is as follows:

5.1 Automatic calculation of the translation between images B and C,which includes:

5.1.1 Automatic detection of holes 2 by image-analysis methods(thresholding) on images B and C.

5.1.2 Calculation of the barycenters of the holes 2 of image C.

5.1.3 Calculation of the barycenters of the hole or holes 2 of image B.

5.1.4 Superposition of the corresponding barycenters.

5.1.5 Calculation of the translation necessary between the two images bycalculating the distance of the two barycenters on the respectiveimages.

5.2 Calculation of Rotation and Deformation (See FIG. 5)

These two functions are to be applied to the image A in relation to thecorresponding image of the preceding specimen section. They makepossible virtual automatic recording of the series of images A.

5.2.1 Calculation of Rotation

The rotation is calculated by the angular sum of rotation related to thefunctioning of the specific apparatus at each magnification and of therotation related to the random orientation of the section on the slide 3of the microscope. The function related to the apparatus is given inadvance; that of the position of the section is calculated on the basisof the orientation of the holes 2 in the image C.

5.2.2 Calculation of the Deformation

Knowing the position of the holes 2 before the section and theirrespective distances, calculation of the inverse deformation to beapplied to the successive images in order to record the holes 2 andconsequently the images that are coordinated with them uses a matrixmodel already tested in prior phases which up to now remains sufficient,namely:

an affine transformation of the first order with 6 parameters.

6. "Recording"

An image-recording step is obtained by digital processing applying tothe images A the series of transformation functions as well as therotations and translations calculated by the process in the inventiondescribed in the preceding step 5.

The process according to the invention is particularly adapted to thereconstruction of the information in three dimensions from successivesections 4 observed with the electron transmission microscope or anoptical microscope. The information can then be used by employingdigital image-processing techniques.

The process according to the invention makes it possible to automate thesearch for areas of one section on another within successive sectionsobserved with the help of electron transmission microscopes whosemovements of the microscope stage and control parameters arecomputer-controlled.

Moreover, the process according to the invention makes is possible toautomate the search for areas of one section on another withinsuccessive sections observed with the aid of optical microscopes whosemovements of microscope stage and control parameters arecomputer-controlled.

We claim:
 1. A process for automating the matching of successivesections of a specimen to be microscopically observed, the processcomprising:providing a marking means; using the marking means to markthe specimen with at least three holes in known relative positionsproximate to an area to be studied; providing a specimen sectioningmeans; using the specimen sectioning means to section said specimen intoa plurality of sections; creating at least three images of each section,a first image being of the area to be studied, a second image being at aweaker magnification than the first image and including same and atleast one of the three holes, and a third image being at the samemagnification as the second image and including all of the holes;digitizing said at least three images; calculating a barycenter for eachhole included in said second and third images; calculating the relativepositions of the barycenters for the holes of the third image;calculating an inverse transformation function from said known relativepositions proximate to the area to be studied and said calculatedrelative positions; normalizing each section first image in contrast andgrey level distribution; applying respective inverse transformationfunctions to each section first image to produce a corrected image foreach section; and producing matched images of the successive sectionsfrom said corrected images.
 2. A process according to claim 1, furthercomprising using said matched images to direct a microscopic imagingdevice to examine a particular area of successive sections.
 3. A methodaccording to claim 1, wherein the marking means comprises a laser.
 4. Amethod according to claim 3, wherein the specimen sectioning meanscomprises a microtome.
 5. A method according to claim 3, wherein thespecimen sectioning means comprises an ultramicrotome.
 6. A methodaccording to claim 2, wherein the marking means comprises a laser.
 7. Amethod according to claim 5, wherein the specimen sectioning meanscomprises a microtome.
 8. A method according to claim 3, wherein thespecimen sectioning means comprises an ultramicrotome.